Mixtures of optical isomers of styralyl alcohol or styralyl acetate, processes for preparing same and organoleptic uses thereof

ABSTRACT

Described are novel mixtures of optical isomers of (i) styralyl alcohol (α-phenylethyl alcohol) or (ii) styralyl acetate (α-phenylethyl acetate), fermentation processes for preparing same and organoleptic uses for same as aroma or food flavor ingredients.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to novel styralyl alcohol isomermixtures and novel sytralyl acetate isomer mixtures, fermentationprocesses for preparing same and organoleptic uses for same as aroma orfood flavor ingredients.

[0002] In today's market, it is frequently desirable to identify flavorcomponents of food items as being “natural flavors.” It is generallyrecognized in the industry that a flavor compound having been preparedby microbial processes can be designated as a natural product andtherefore have an important place in the commercialization of productscontaining them. As a result, the industry has devoted considerable timeand effort to develop methods for the production of flavoring componentsand, in particular, for the production of certain alcohol and acetatederivatives which can properly be called “natural.”

[0003] Furthermore, in today's market, a trend is developing whereby itis now determined to be desirable to identify fragrance ingredients offragrance compositions as being “natural fragrance ingredients.” It isgenerally recognized in the industry that a fragrance compound havingbeen prepared by microbial processes can be designated as a naturalproduct and therefore have an important place in the commercializationof perfumes and perfumed articles containing them. As a result, theindustry has devoted considerable time and effort to develop methods forthe production of fragrance components and in particular for theproduction of alcohols and esters which can be called “natural fragranceingredients.”

[0004] It is also well known in the flavor and fragrance industry thatparticular stereoisomers of specific ingredients are, in many instances,more advantageous than their corresponding opposite stereoisomers.Accordingly, a significant amount of research has been carried outcovering the formation of such stereoisomers using, for example,microbial reduction.

[0005] Thus, Simon, et ad, Angew. Chem. Int. Ed Engl. 24 (1985), pages539-553 (title: “Chiral Compounds Synthesized by BiocatalyticReductions”) discloses at section 3.2 on page 545 the hydrogenation ofketones to form chiral secondary alcohols-using Clostridia such asClostridium kluyveri and specifically sets forth the production ofα-phenylethyl alcohol stereoisomers having the structure:

[0006] with an enantiomeric excess of 94%.

[0007] Adlercreutz, Biotechnology Letters, Volume 13, No. 4 atpages-229-234 (1991) (title: “ASYMMETRIC REDUCTION OF KETONES WITHENZYMES FROM ACETIC ACID BACTERIA”) shows production of α-phenylethylalcohol at page 233 using G. oxydans (an enantiomeric excess of 99%); A.aceti (an enantiomeric excess of 92%); G. oxydans (an enantiomericexcess of 75%); A. aceti (an enantiomeric excess of 86%); A.pasteurianus (an enantiomeric excess of 94%); and A. peroxydans (anenantiomeric excess of 66%).

[0008] Nakamura, et al, Tetrahedron: Asymmetry, Volume 7, No. 10 atpages 3021-3024, 1996, published by Elsevier Science Ltd. (title:“Asymmetric Synthesis of (S)-Arylalkanols by Microbial Reduction”) setsforth the production of the isomers having the structures:

[0009] using acetophenone as a starting material in accordance with thereaction:

[0010] using Geoirichum candidum IFO 4597, but only shows the productionof a stereoisomer mixture having 28% enantiomeric excess.

[0011] Vicenzi, et al, Enzyme and Microbial Technology, 20: pages494-499, 1997, published by Elsevier Science Inc. (title: “Large-scalestereoselective enzymatic ketone reduction with in situ product removalvia polymeric adsorbent resins”) discloses the stereoselective enzymaticreduction of 3,4-methylene-dioxyphenyl acetone to the correspondingS-3,4-methylene-dioxyphenyl isopropanol utilizing Zygosaccharomycesrouxii.

[0012] Sorrilha, et al, Organic & Medicinal Chemistry Letters, Volume 2,No. 2 at pages 191-196, 1992, published by Pergamon Press plc (title:“REDUCTION OF PHENYLKETONES BY IMMOBLIZED BAKER'S YEAST”) discloses aprocess wherein baker's yeast immobilized on chrysotile andmontmorillonite causes stereoselective reduction of1-phenyl-1,2-propanedione to the corresponding (1R,2S)-diol.

[0013] Nothing in the prior art, however, discloses the production ofthe stereoisomeric mixture of the compounds having the structures:

[0014] having an α_(D) ²⁰=−38.6° with an enantiomeric excess percent of87.5 ([∈∈%=87.5]) and furthermore, nothing in the prior alt disclosesthe production of the stereoisomeric mixture of esters having thestructures:

[0015] having (α_(D) ²⁰=+73.06°) with an enantiomeric excess percent of79.2, [∈∈%=79.2] or α_(D) ²⁰=−102.7° with an enantiomeric excess percentof 89.15, [∈∈%=89.15], which materials have been found by us to haveunexpected, unobvious and advantageous properties insofar as theirorganoleptic (flavor and fragrance) utilities are concerned.

SUMMARY OF THE INVENTION

[0016] The above and other objects and features of the invention areobtained in accordance with the present invention by carrying out aprocess using (i) reductive reaction techniques to produce and recovercertain naturally occurring alcohols and (ii) esterification reactiontechniques to produce and recover certain naturally occurring esters,found to be useful for their organoleptic properties in augmenting orenhancing the aroma or taste of consumable materials such as foodstuffs,chewing gums, toothpastes, oral care products, chewing tobaccos, smokingtobaccos, perfume compositions, colognes and perfumed articles such assolid or liquid detergents, perfumed polymers, fabric softenercompositions, fabric softener articles, cosmetic powders, hairpreparations and the like.

[0017] The reductive reaction products are styralyl alcohol stereoisomermixtures containing compounds having the structures:

[0018] produced according to a fermentation reaction wherebyacetophenone is reduced according to the reaction:

[0019] using a culture of Kluyveromyces polysporus ATCC 22028.

[0020] The resulting products may be used as is for their organolepticproperties or may be further reacted by means of esterification withacetic acid according to the reaction:

[0021] The aforementioned esterification reaction may take place usingsuch esterification catalysts as citric acid or may take place by meansof a fermentation reaction using an ester-forming enzyme, preferablyCandida antarctica esterase expressed in Asperigilus orzae, for exampleNOVOZYM® 435, a triacylglycerol hydrolase (E.C. No. 3.1.1.3) acting asan effective carboxylesterase. NOVOZYM® is a trademark of the NovoNordisk A/S Organization of Novo Alle, 2880 Bagsvaerd, Denmark.

[0022] When carrying out the reductive fermentation reaction, to wit:

[0023] the resulting product has an optical rotation of −38.6°,[α=−38.6°] and an enantiomeric excess percent of about 87.5,[∈∈%=87.5±0.5].

[0024] When carrying out the esterification reaction, to wit:

[0025] using a citric acid catalyst, the resultant stereoisomer mixturehas an optical activity of −102.7°, [α=−102.7°] with an enantiomericexcess percent of about 78.31, [∈∈%=78.31±0.5].

[0026] When carrying out the esterification reaction, to wit:

[0027] using the enzyme Candida antarctica esterase expressed inAsperigilus orzae, specifically NOVOZYM® 435, a triacylglycerolhydrolase (E.C. No. 3.1.1.3) acting as an effective carboxylesterase, amixture of stereoisomers having an optical activity of +73.06° isproduced ([α=+73.06°]) with an enantiomeric excess percent of about81.1, [∈∈%=81.1±0.5].

[0028] The mixture of optical isomers of the styralyl alcohol having thestructures:

[0029] produced as set forth above having an optical rotation of −38.6°,[α=−38.6°] with an enantiomeric excess percent of about 87.5,[∈∈%=87.5±0.5] has a mild hyacinth, gardenia aroma with strawberrynuances. It is, accordingly, useful in augmenting enhancing andimparting aroma in or to floral fragrances and is useful in the creationof strawberry-flavored foodstuffs; e.g., strawberry-flavored gelatindesserts, strawberry-flavored chewing gums and strawberry-flavored fruitpreparations for yogurt.

[0030] The mixture of stereoisomers of esters having the structures:

[0031] prepared as set forth above using a citric acid catalyst andhaving an optical rotation of −102.7°, [α=−102.7°] and an enantiomericexcess percent of about 78.31, [∈∈%=78.31±0.5] has a fresh strawberry,green, dried fruit aroma with a green, avocado taste profile andstrawberry jam nuances. From a fragrance standpoint, this substance isdescribed as having a strawberry, green, dried fruit aroma withstrawberry topnotes.

[0032] The mixture of stereoisomeric esters having the structures:

[0033] prepared as set forth above using the ester-forming enzyme,Candida antarctica esterase expressed in Asperigilus orzae, and havingan optical rotation of +73.06°, [α=+73.06°] with an enantiomeric excesspercent of about 81.1, [∈∈%=81.1±0.5] has a fruity, floral, jasmine,mimosa, gardenia aroma with apricot, apple and strawberry jam flavornuances. This material is also useful in the formation of fruity,floral, jasmine fragrances, as well as strawberry-flavored foodstuffsand food preparations such as strawberry-flavored gelatin desserts,strawberry-flavored ice cream and strawberry-flavored preparations foryogurts.

[0034] In carrying out the process for production of the styralylalcohol stereoisomer mixtures containing stereoisomers having thestructures:

[0035] an inoculum preparation is first prepared containing a culture ofKluyveromyces polysporus ATCC 22028 and also containing nutrientsincluding dextrose. The inoculum, after incubation, is then placed in aproduction fermenter which is aerated and agitated at, for example, anaeration rate of 0.25 v/v/m; at a temperature of, for example, 25° C.;and at an agitation rate of, for example, 300 rpm for a time period of,for example, 24 hours. As set forth in the Detailed Description of theInvention, infra, the pH range may vary from about 5.5 up to about 6.

[0036] The resulting product is then extracted with a solvent such asethyl acetate, and the solvent extract is then washed in order to bringthe pH up to approximately neutral (pH=7). The resulting washed extractis then concentrated and the resulting concentrate is then fractionallydistilled. The resulting distillate is then used “as is” for itsorganoleptic properties or further reacted with acetic acid according tothe reaction:

[0037] When carrying out the reaction using a citric acid catalyst, theresulting distillate is admixed with acetic acid and citric acid, andthe resulting mixture is refluxed for about 15 hours while excess aceticacid is recovered. At the end of the 15 hour period, the resultingproduct is fractionally distilled.

[0038] When carrying out the esterification reaction, to wit:

[0039] using the ester-forming enzyme, Candida antarctica esteraseexpressed in Asperigilus orzae, specifically, NOVOZYM® 435, the mixtureof styralyl alcohol stereoisomers having the structures:

[0040] is admixed with a small amount of water and a small amount ofNOVOZYM® 435. The resulting mixture is heated to 40° C. and acetic acidis then added over a period of between about 5 and about 10 hours andthen continuously stirred for an additional 40-60 hours. The conversionusing the “enzyme” process is between about 2.5 and about 3.5%. Theresulting product is then fractionally distilled at a pressure of 5mm/Hg. and at a head temperature of 34-38° C.

BRIEF DESCRIPTION OF TIHE DRAWINGS

[0041] The present invention will be further understood with referenceto the accompanying drawings wherein:

[0042]FIG. 1A is a GC-mass spectrum for the reaction product for ExampleI containing the mixture of stereoisomers having the structures:

[0043]FIG. 1B is another GC-mass spectrum for the mixture ofstereoisomers having the structures:

[0044] produced according to Example 1.

[0045]FIG. 1C is a GLC profile for the reaction product of Example Icontaining a mixture of stereoisomers having the structures:

[0046]FIG. 1D is a mass spectrum for the product of Example I containinga mixture of stereoisomers having the structures:

[0047]FIG. 1E is a GLC profile for the reaction product of Example Icontaining the stereoisomers having the structures:

[0048]FIG. 1F is a mass spectrum for the reaction product of Example Icontaining the stereoisomers having the structures:

[0049]FIG. 2A is a GLC profile for the reaction product of Example IIcontaining the stereoisomers having the structures:

[0050]FIG. 2B is a “history plot” for the product formed according tothe process of Example II which contains and consists essentially of thestereoisomers having the structures:

[0051]FIG. 2C is a GC-mass spectrum for the reaction product of ExampleII containing the stereoisomers having the structures:

[0052]FIG. 2D is a CC-mass spectrum for the reaction product of ExampleII containing the stereoisomers having the structures:

[0053]FIG. 3A is a chiral chromatogram for the reaction product ofExample IV containing the stereoisomers having the structures:

[0054] with the optical rotation of +73.06°, [α=+73.06°] and with anenantiomeric excess percent of about 81.1, [∈∈%=81.1±0.5].

[0055]FIG. 3B is another chiral GC chromatogram for the reaction productof Example IV containing a mixture of stereoisomers having thestructures:

[0056] with an optical rotation of +73.06°, [α=+73.06°] and having anenantiomeric excess percent of about 81.1, [∈∈%=81.1±0.5].

[0057]FIG. 3C is a GLC profile for the reaction product of Example IVcontaining stereoisomers having the structures:

[0058]FIG. 4 represents a cutaway side elevation view of apparatus usedin forming perfumed polymers which contain embedded therein at least oneof the stereoisomers having one of the structures:

[0059]FIG. 5 is a front view of the apparatus of FIG. 4 looking in thedirection of the arrows along lines 5-5.

[0060]FIG. 6A is a GC-mass spectrum (using a Carbowax column) for thereaction product of Example III containing a mixture of stereoisomershaving the structures:

[0061] with the mixture having an optical rotation of −102.7°,[α=102.7°] and having an enantiomeric excess percent of about 78.31,[∈∈%=78.31±0.5.

[0062]FIG. 6B is a GC-mass spectrum for the reaction product of ExampleIII (using an OV-1 column) containing the stereoisomers having thestructures:

[0063] with the mixture having an optical rotation of −102.7°,[α=−102.7°] and an enantiomeric excess percent of about 78.31,[∈∈%=78.31±0.5].

DETAILED DESCRIPTION OF THE DRAWINGS

[0064] Referring to FIG. 1D, the peak indicated by reference numeral 10is the peak for acetic acid. The peak indicated by reference numeral 11is the peak for ethyl acetate. The peak indicated by reference numeral12 is for isobutyl alcohol. The peak indicated by reference numeral 13is for 3-methyl-butanol. The peak indicated by reference numeral 14 isfor a mixture of acetophenone and the mixture of stereoisomers havingthe structures:

[0065] The peak indicated by reference numeral 15 is for phenylethylalcohol having the structure:

[0066] The peak indicated by reference numeral 16 is for the mixture ofstereoisomers having the structures:

[0067] The peak indicated by reference numeral 17 is the peak foracetophenone. The peak indicated by reference numeral 18 is for thecompound having the structure:

[0068] The peak indicated by reference numeral 19 is the peak forβ-phenylethyl acetate having the structure:

[0069] Referring to FIG. 1F, the peak indicated by reference numeral 20is the peak for ethyl acetate. The peak indicated by reference numeral21 is for isobutyl alcohol. The peak indicated by reference numeral 22is for 3-methyl-butanol. The peak indicated by reference numeral 23 isfor acetic acid. The peak indicated by reference numeral 24 is for2-methyl-propionic acid. The peak indicated by reference numeral 25 isfor acetophenone. The peak indicated by reference numeral 26 is for themixture of stereoisomers having the structures:

[0070] The peak indicated by reference numeral 27 is the peak forβ-phenylethyl alcohol having the structure:

[0071] Referring to FIG. 3A, the peaks indicated by reference numerals31 and 32 are for the stereoisomers having the structures:

[0072] The peak indicated by reference numeral 30 is for the mixture ofstereoisomers having the structures:

[0073] With reference to FIG. 3B, the peaks indicated by referencenumerals 34 and 35 are for the stereoisomers having the structures:

[0074] separately. The peak indicated by reference numeral 33 is for themixture of stereoisomers having the structures:

[0075] With reference to FIG. 3C, the peak indicated by referencenumeral 36 is the peak for the mixture of stereoisomers having thestructures:

[0076] The peak indicated by reference numeral 37 is the peak for themixture of stereoisomers having the structures:

[0077] Referring to FIGS. 4 and 5, there is provided a process forforming scented polymer elements (wherein the polymer may be athermoplastic polymer such as low density polyethylene or polypropyleneor copolymers of ethylene and vinyl acetate or mixtures of polymers andcopolymers such as copolymers of ethylene and vinyl acetate andpolyethylene) such as pellets useful in the formation of plasticparticles useful in fabricating certain articles which may be perfumed.This process comprises heating the polymer or mixture of polymers to themelting point of said polymer or mixture of polymers, e.g., 250° C. inthe case of low density polyethylene. The lower most portion of thecontainer is maintained at a slightly lower temperature, and thematerial in the container is taken off at such location for deliverythrough the conduit (indicated by reference numeral 218). Thus,referring to FIGS. 4 and 5 in particular, the apparatus used inproducing such elements comprises a device for forming the polymercontaining perfume, e.g., polyethylene or polyethylene-polyvinyl acetateor mixtures of same or polypropylene, which comprises a vat or container212 into which the polymer taken alone or in admixture with othercopolymers and the perfuming substance, which is at least one of thestereoisomers having one of the structures:

[0078] of our invention and other compatible perfumes, is placed. Thecontainer is closed by means of an airtight lid 228 and clamped to thecontainer by bolts 265. A stirrer 273 traverses the lid or cover 228 inan airtight manner and is rotatable in a suitable manner. A surroundingcylinder 212A having heating coils which are supplied with electriccurrent through cable 214 from a rheostat or control 216 is operated tomaintain the temperature inside the container 212 such that the polymerin the container will be maintained in the molten or liquid state. Ithas been found advantageous to employ polymers at such a temperaturethat the viscosity will be in the range of 90-100 Saybolt seconds. Theheater is operated to maintain the upper portion of the container 212within a temperature range of, for example, 220°-270° C. in the case oflow density polyethylene. The bottom portion of the container 212 ishealed by means of heating coils 212A regulated through the control 220connected thereto through a connecting wire 222 to maintain the lowerportion of the container 212 within a temperature range of 220°-270° C.

[0079] Thus, the polymer or mixture of polymers added to the container212 is heated from 10-12 hours, whereafter the perfume composition orperfume material which contains one or more of the stereoisomers havingthe structures:

[0080] of our invention is quickly added to the melt. Generally, about10-45% by weight of the resulting mixture of the perfumery substance isadded to the polymer.

[0081] After the perfume material is added to the container 212, themixture is stirred for a few minutes, for example, 5-15 minutes, andmaintained within the temperature ranges indicated previously by theheating coils 212A. The controls 216 and 220 are connected throughcables 224 and 226 to a suitable supply of electric current forsupplying the power for heating purposes.

[0082] Thereafter, the valve “V” is opened permitting the mass to flowoutwardly through conduit 232 (also shown by reference numeral 218 (incutaway cross section)) having a multiplicity of orifices 234 adjacentto the lower side thereof. The outer end of the conduit 232 is closed sothat the liquid polymer in intimate admixture with one or more of thestereoisomers having one of the structures:

[0083] and one or more other substances (if desired) will continuouslydrop through orifices 234 downwardly from the conduit 232. During thistime, the temperature of the polymer intimately admixed with theperfumery substance in the container 212 is accurate], controlled sothat a temperature in the range of form about 240°-250° C., for example(in the case of low density polyethylene) will exist in the conduit 232.The regulation of the temperature through the controls 216 and 220 isessential in order to insure temperature balance to provide for thecontinuous dripping or dropping of molten polymer intimately admixedwith the perfume substance which contains one or more of thestereoisomers having one of the structures:

[0084] of our invention, through the orifices 234 at a rate which willinsure the formation of droplets 236 which will fall downwardly onto amoving conveyor belt 238 caused to run between conveyor wheels 240 and242 beneath the conduit 232.

[0085] When the droplets 236 fall onto the conveyor 238, they formpellets 244 which harden almost instantaneously and fall off the end ofthe conveyor 238 into a container 245 which is advantageously filledwith water 252 or some other suitable cooling liquid to insure the rapidcooling of each of the pellets 244. The pellets 244 are then collectedfrom the container 245 and utilized for the formation of otherfunctional products, e.g., garbage bags and the like.

[0086] A feature of the invention is the provision for the moistening ofthe conveyor belt 238 to insure the rapid formation of the solidpolymer-aromatizing agent containing pellets 244 without sticking to thebelt. The belt 238 is advantageously of a material which will notnormally stick to a melted polymer, but the moistening means 248 insuresa sufficiently cold temperature of the belt surface for the adequateformation of the pellets 244. The moistening means comprises a container250 which is continuously fed with water 252 to maintain a level 254 formoistening a sponge element 256 which bears against the exterior surfaceof the belt 238.

DETAILED DESCRIPTION OF THE INVENTION

[0087] The reactions according to the present invention are shownthusly:

[0088] More specifically, the first reaction involving reduction of aketone group to form a secondary alcohol is carried out in the presenceof Kluyveromyces polysporus ATCC 22028.

[0089] The process is carried out by first introducing an inoculumcontaining the said Kluyveromyces polysporus ATCC 22028 into a reactionvessel which contains a production medium, typically including anutrient source, a buffering agent such as KH₂PO₄, a yeast extract andother nutrient sources which may include trace minerals and growthfactors.

[0090] Immediately, subsequent to the introduction of the inoculum, asource of dextrose in a suitable nutrient medium is begun feeding intothe reaction vessel (and throughout the entire fermentation procedure).

[0091] Up to about 30 hours after inoculation, acetophenone having thestructure:

[0092] is pumped into the reaction vessel. Incubation is then continuedat a temperature in the range of 20° C. up to 30° C., at an agitationrate of from about 250 up to about 350 rpm and an aeration rate of fromabout 0.20 up to about 0.40 v/v/m. Conversion to the resulting mixtureof stereoisomers having the structures:

[0093] is monitored by means of GC.

[0094] At the conclusion of the fermentation, the fermentation broth ispasteurized for a period of between about 30 up to about 70 minutes at atemperature of between about 60° C. and about 75° C., cooled and stored.The crude product is recovered by extraction of the whole broth using anequal volume of a suitable solvent, preferably ethyl acetate. The spentbroth is extracted a second time and the recovered ethyl acetate layersare combined and washed with aqueous sodium chloride, preferably. Thesolvent is then evaporated using such equipment as a rotary evaporatorat a water bath temperature in the range of from about 35° C. up toabout 50° C. under vacuum. The crude extract is then further purified,if desired, for use for its organoleptic properties; or, in thealternative, the crude extract may be converted to styralyl acetatedirectly without any further purification; or, optionally, the resultingdistillate can be converted to styralyl acetate.

[0095] Using the aforementioned procedure, the optical rotation of theresulting mixture of stereoisomers having the structures:

[0096] is −38.6° with an enantiomeric excess percent of about 87.5,[∈∈%=87.5±0.5]. The aforementioned term is intended to mean that theenantiomeric excess percent is between 87 and 88%.

[0097] If desired, the resulting mixture of stereoisomers having thestructures:

[0098] may further be reacted with acetic acid in order to form theresulting esters having the structures:

[0099] As stated, supra, and as exemplified, infra, the aforementionedesters may be formed, in the alternative, into a mixture ofstereoisomers having an optical rotation of −102.7°, [α=−102.7°] with anenantiomeric excess percent of about 78.31, [∈∈%=78.31±0.5] whencarrying out the esterification with a citric acid catalyst or themixture of esters may have an optical rotation of +73.06° with anenantiomeric excess percent of about 81.1, [∈∈%=81.1±0.5] when using anester-forming enzyme which is Candida antarctica esterase expressed inAsperigilus orzae, preferably NOVOZYM® 435, a triacylglycerol hydrolase(E.C. No. 3.1.1.3) acting as an effective carboxylesterase.

[0100] When carrying out the reaction:

[0101] using a citric acid catalyst, excess acetic acid is admixed withthe stereoisomers having the structures:

[0102] and a small amount of citric acid, e.g., approximately 0.1% ofthe weight of the entire reaction mass. The resulting mixture is thenrefluxed for a period of time of between about 10 up to about 30 hours.During such refluxing, a substantial quantity of the excess acetic acidis recovered by means of azeotropic distillation with water of reaction.The reaction is carried out at a reflux temperature of approximately115-120° C. at atmospheric pressure.

[0103] At the end of the reaction, the reaction product is fractionallydistilled to yield a mixture of isomers having the structures:

[0104] wherein the mixture has an optical rotation of −102.7°,[α=−102.7°] with an enantiomeric excess percent of about 78.31,[∈∈%=78.31±0.5].

[0105] In the alternative, in place of the citric acid catalyst, afermentation esterification can be carried out using the ester-formingenzyme, Candida antarctica esterase expressed in Asperigilus orzae,preferably NOVOZYM® 435, a triacylglycerol hydrolase (E.C. No. 3.1.1.3)acting as an effective carboxylesterase.

[0106] In carrying out the reaction using the ester-forming enzyme, thereaction may be carried out with the ester-forming enzyme, the aceticacid and the styralyl alcohol with a small amount of water, or thereaction may be carried out in the presence of an inert solvent such ashexane. We have found that when using the inert solvent such as hexane,a higher conversion is achieved. Thus, initially, styralyl alcohol,water and the ester-forming catalyst are initially admixed and theresulting mixture is heated to a temperature of between about 35° C. and45° C. The resulting mixture is then further admixed with acetic acidwhich is added over a period of between about 5 and 10 hours whilemaintaining the reaction mass at 40° C. Optionally, solvents such ashexane may be introduced into the mixture of styralyl alcohol and waterprior to the introduction of acetic acid. When a solvent such as hexaneis utilized, the conversion of the styralyl alcohol to the ester isbetween about 7 and about 10%. However, when such solvent is not used,the conversion is between about 2.5 up to about 3.5%.

[0107] Accordingly, it is preferred that a solvent such as n-hexane beutilized in carrying out the esterification reaction using anester-forming enzyme. The amount of such solvent as n-hexane isapproximately equal to the weight of styralyl alcohol in the reactionmass.

[0108] The mixtures of stereoisomers of styralyl alcohol and styralylacetate obtained in accordance with the present invention and one ormore auxiliary perfume ingredients including, for example, hydrocarbons,alcohols other than the styralyl alcohols of out invention, ketones,aldehydes, nitriles, esters other than the styralyl acetates of ourinvention, ethers, synthetic essential oils, lactones and naturalessential oils may be admixed so that the combined odors of theindividual components produce a pleasant and desired fragrance,particularly and preferably in the fruity and floral area (e.g., jasmineand gardenia aromas). Such perfume compositions usually contain (a) themain note or the “bouquet” or foundation stone of the composition; (b)modifiers which round off and accompany the main note; (c) fixativeswhich include odorous substances which lend a particular note to theperfume throughout all stages of evaporation and substance which retardevaporation; and (d) topnotes which are usually low boiling,fresh-smelling materials.

[0109] In perfume compositions, it is the individual compositions whichcontribute to their particular olfactory characteristics; however, theoverall sensory effect of the perfume compositions will be at least thesum total of the effects of each of the ingredients. Thus, one or moreof the styralyl acetate or styralyl alcohol stereoisomer mixtures of ourinvention can be used to alter, modify or enhance the aromacharacteristics of a perfume composition, for example, by utilizing ormoderating the olfactory reaction contributed by another ingredient inthe composition.

[0110] The amount of styralyl alcohol or styralyl acetate stereoisomercomposition of our invention, which will be effective in perfumecompositions as well as in perfumed articles and colognes, depends uponmany factors including the other ingredients, their amounts and theeffects which are desired. It has been found that perfume compositionscontaining as little as 0.005% of the styralyl alcohol or styralylacetate stereoisomers or even less (e.g., 0.0025%) can be used to impartgreen, floral, hyacinth, gardenia, jasmine, mimosa, fruity, dried fruitand strawberry aromas with green, fruity, strawberry, “fresh air-driedclothing” topnotes to soaps, cosmetics, detergents including anionic,cationic, nonionic and zwitterionic solid or liquid detergents, perfumedpolymers and other products. The amount employed can range up to 70% ofthe fragrance and will depend upon consideration of cost, nature of theend product, the effect desired on the finished product and theparticular fragrance sought.

[0111] The mixtures of styralyl alcohol stereoisomers and styralylacetate stereoisomers of this invention are useful when taken alone ortaken together with other perfumery ingredients in detergents, soaps,space ordorants and deodorants, perfumes, colognes, toilet waters, bathpreparations, hair preparations such as lacquers, brilliantines,pomades, shampoos; cosmetic preparations such as creams, deodorants,hand lotions and sun screens; powder such as talcs, dusting powders,face powders and the like.

[0112] As little as 0.25% of the mixtures of styralyl alcoholstereoisomers or styralyl acetate stereoisomers can suffice to impartintense and substantive green, floral, hyacinth, gardenia jasmine,mimosa, fruity, dried fruit and strawberry aromas with green, fruity,strawberry and “fresh air-dried clothing” topnotes to floral and fruityperfume formulations. Generally, no more than 5% of the mixtures ofstyralyl alcohol stereoisomers or styralyl acetate stereoisomers basedon the ultimate end product is required to be used in the perfumecompositions.

[0113] Furthermore, as little as 0.25% of the mixture of styralylalcohol stereoisomers or styralyl acetate stereoisomers can suffice toimpart such aromas to perfumed articles, per se, whether in the presenceof other perfume materials or whether used by themselves. Thus, therange of use of the mixtures of styralyl alcohol stereoisomers orstyralyl acetate stereoisomers of this invention in perfumed articles,e.g., perfumed polymers and solid or liquid anionic, cationic, nonionicor zwitterionic solid or liquid detergents may vary from 0.25% up toabout 5% by weight based on the total weight of the perfumed article.

[0114] In addition, the perfume composition or fragrance composition ofour invention can contain a vehicle or carrier for the mixture ofstyralyl alcohol stereoisomers or styralyl acetate stereoisomers of ourinvention. The vehicle can be a liquid such as a nontoxic alcohol (e.g.,ethanol); a nontoxic glycol (e.g., propylene glycol) or the like. Thecarrier can also be an absorbent solid such as a gum (e.g., gum arabic,xanthan gum or guar gum) or components for encapsulating the compositionby means of coacervation (such as by gelatin) or by means of theformation of a polymer around a liquid center. This can be accomplishedby using a urea formaldehyde prepolymer to form a polymeric capsulearound a liquid perfume composition center as is known in the prior art.

[0115] It will be appreciated from the present disclosure that themixtures of styralyl alcohol stereoisomers or styralyl acetatestereoisomers according to the present invention can be used to alter,vary, fortify, modify, enhance or otherwise improve the flavor of a widevariety of materials which are ingested, consumed or otherwiseorganoleptically sensed.

[0116] The terms “alter” and “modify” in their various forms will beunderstood herein to mean the supplying or imparting of a flavorcharacter or note to an otherwise bland, relatively tasteless substanceor augmenting an existing flavor characteristic where the natural flavoris deficient in some regard or supplement the existing flavor impressionto modify its organoleptic character.

[0117] The term “enhance” is intended herein to mean the intensification(by use of the mixtures of styralyl alcohol stereoisomers or styralylacetate stereoisomers of this invention) of a flavor, aroma note ornuance in a foodstuff, perfume composition or perfumed article withoutchanging the quality of said note or nuance.

[0118] A “flavoring composition” as referred to herein means one whichcontributes a part of the overall flavor impression by supplementing orfortifying a natural or artificial flavor in a material or one whichsupplies substantially all the flavor and/or aroma character to aconsumable article.

[0119] The term “foodstuff” as used herein includes both solid andliquid ingestible material for man or animals, which materials usuallydo, but need not have nutritional value. Thus, foodstuffs include meats;gravies; soups; convenience foods; malt; alcoholic and other beverages;milk and dairy products; seafood including fish, crustaceans, mollusksand the like; candies; vegetables; cereals; soft drinks; snacks; dog andcat foods; other veterinary products and the like.

[0120] When the mixtures of styralyl alcohol stereoisomers or styralylacetate stereoisomers of this invention are used in a flavoringcomposition, they can be combined with conventional flavoring materialsor adjuvants. Such co-ingredients or flavoring adjuvants are well knownin the art for such and have been extensively described in theliterature. Requirements of such adjuvant materials are:

[0121] (1) that they be nonreactive with the styralyl alcoholstereoisomer mixtures or styralyl acetate stereoisomer mixtures of thisinvention;

[0122] (2) that they be organoleptically compatible with the mixtures ofstyralyl alcohol stereoisomers or styralyl acetate stereoisomers of thisinvention whereby the flavor of the ultimate consumable material towhich the styralyl alcohol stereoisomers or styralyl acetatestereoisomers are added is not detrimentally affected by the use of theadjuvant; and

[0123] (3) that they be ingestible, acceptable and thus nontoxic orotherwise nondeleterious.

[0124] Apart from these requirements, conventional materials can be usedand broadly include other flavor materials, vehicles, stabilizers,thickeners, surface active agents, conditioners and flavor intensifiers.

[0125] Such conventional flavoring materials include saturated fattyacids; unsaturated fatty acids and amino acids; alcohols (other than thestyralyl alcohol stereoisomers of our invention) including primary andsecondary alcohols; esters (other than the styralyl acetate stereoisomermixtures of our invention); carbonyl compounds including ketones andaldehydes; lactones; other cyclic organic materials including benzenederivatives, alicyclic compounds, heterocyclics such as furans,pyridines, pyrazines and the like; sulfur-containing compounds includingthiols, sulfides, disulfides and the like; proteins; lipids;carbohydrates; so-called flavor potentiators such as monosodiumglutamate, magnesium glutamate, calcium glutamate, guanylates, andinosinates; natural flavoring materials such as cocoa, vanilla andcaramel; essential oils and extracts such as anise oil, clove oil andthe like; and artificial flavoring materials such as vanillin and thelike.

[0126] Specific preferred flavor adjuvants are as follows:

[0127] anise oil;

[0128] ethyl-2-methyl butyrate;

[0129] vanillin;

[0130] cis-3-heptenol;

[0131] cis-3-hexenol;

[0132] trans-2-heptenol;

[0133] cis-3-heptenal;

[0134] butyl valerate;

[0135] 2,3-diethyl pyrazine;

[0136] methyl cyclopentenolone;

[0137] benzaldehyde;

[0138] valerian oil;

[0139] 3,4-dimethoxyphenol;

[0140] amyl acetate;

[0141] amyl cinnamate;

[0142] γ-butyryl lactone;

[0143] trimethyl pyrazine;

[0144] phenyl acetic acid;

[0145] isovaleraldehyde;

[0146] ethyl maltol;

[0147] ethyl vanillin;

[0148] ethyl valerate;

[0149] cocoa extract;

[0150] coffee extract;

[0151] peppermint oil;

[0152] spearmint oil;

[0153] clove oil;

[0154] anethol;

[0155] cardamom oil;

[0156] wintergreen oil;

[0157] cinnamic aldehyde;

[0158] ethyl-2-methyl valerate;

[0159] γ-hexenyl lactone;

[0160] 2,4-decadienal;

[0161] 2,4-heptadienal;

[0162] 2-methyl-2-pentenoic acid;

[0163] 2-methyl-3-pentenoic acid;

[0164] the ethyl ester of 2-methyl-2-pentenoic acid;

[0165] the ethyl ester of 2-methyl-3-pentenoic acid;

[0166] the ethyl ester of 2-methyl-4-pentenoic acid;

[0167] 2-methyl-4-pentenoic acid;

[0168] the hexyl ester of 2-methyl-3-pentenoic; and

[0169] butylidene phthalide.

[0170] The following table sets forth organoleptic properties of themixtures of styralyl alcohol stereoisomers and styralyl acetatestereoisomers of our invention: TABLE I Description of Composition FruitFlavor Properties Fragrance Properties Mixture of stereoisomers havingthe A strawberry flavor. A hyacinth, gardenia aroma structures: withstrawberry topnotes.

having an optical rotation of −38.6°, [α = −38.6°] and an enantiomericexcess percent of about 87.5, [∈ ∈ % = 87.5 ± 0.5]. Mixture ofstereoisomers having the A strawberry jam, A fruity, floral, jasmine,structures: apricot and apple flavor. mimosa and gardenia aroma withstrawberry, fruity and green topnotes.

having an optical rotation of +73.06°, [α = +73.06°] and an enantiomericexcess percent of about 81.1, [∈ ∈ % = 81.1 ± 0.5]. Mixture ofstereoisomers having the A strawberry jam, green, A strawberry, green,dried structures: dried fruit, avocado taste. fruit aroma withstrawberry, green, “fresh air-dried clothing” topnotes.

having an optical rotation of −102.7°, [α = −102.7°] and an enantiomericexcess percent of about 78.31, [∈ ∈ % = 78.31 ± 0.5].

[0171] The following Examples I, II, III and IV illustrate methods ofour invention used to produce the mixtures of stereoisomers of styralylalcohol and mixtures of stereoisomers of styralyl acetate of ourinvention. Examples V, et seq, serve to illustrate the organolepticutilities of the mixtures of stereoisomers of styralyl alcohol andmixtures of stereoisomers of styralyl acetate of our invention. Allparts and percentages given herein are by weight unless otherwisespecified.

EXAMPLE I Production of Styralyl Alcohol In Laboratory Fermenters

[0172] I. Inoculum Preparation

[0173] A. Medium:

[0174] The following medium was prepared by admixing the followingingredients: g/L Ingredients 5.0 AMBERFERM ® 4000 Peptone 5.0 TAST0NE ®900 5.0 KH₂PO₄ (potassium phosphate, monobasic) 5.0 Malt extract 102 0.5MgSO₄.7H₂O (magnesium sulfate, heptahydrate) 1 L Deionized water 50.0 CERELOSE ® 2001 (added after sterilization) (dextrose)

[0175] B. Parameters: Temperature: 25° C.; Agitation: 100 rpm; andDuration: 24 hours.

[0176] A 2.8 liter Fernbach flask containing 500 ml of the inoculummedium of A, supra, was sterilized at 121° C. for 30 minutes. The flaskwas inoculated with 1.8 ml of a frozen culture of Kluyveromycespolysporus ATCC 22028; 50.0 ml of sterile 50% dextrose (CERELOSE® 2001)and 2-3 drops of antifoam were added. The flask was inoculated in ashaker (100 rpm) at 25° C. for 24 hours.

[0177] II. Production

[0178] A. Medium:

[0179] The following medium was prepared by admixing the followingingredients: g/L Ingredients 50.0 AMBERFERM ® 4000 Peptone 50.0TASTONE ® 900 50.0 Malt extract 102 50.0 KH₂PO₄ (potassium phosphate,monobasic)  5.0 MgSO₄.7H₂O (magnesium sulfate, heptahydrate) 10.0Antifoam Hi MARS-010 ® FGK 11.0 L Deionized water 500.0  CERELOSE ® 2001(added after sterilization) (dextrose)

[0180] B. Fermenter Parameters: Inoculum: 500 ml of propagation culture;Broth Volume: 11.0 liters; Temperature: 25° C.; Aeration: 0.25 v/v/m;Agitation: 300 rpm (impeller diameter 84 mm); and Duration: 24 hours.

[0181] C. Process:

[0182] A total of 10 liters of medium (with an additional 1 liter ofwater added to make up for steam lost during sterilization) wassterilized in a 23 liter fermenter at 121° C. for 30 minutes. Aftersterilization, 1 liter of sterile 50% dextrose (CERELOSE® 2001) and 500ml of a 24 hours-grown inoculum were added. During the fermentation, thesugar was monitored but not adjusted.

[0183] After 15.5 hours of incubation, the dextrose and ethanol weredetermined and the optical density (OD) at 600. of a {fraction (1/100)}dilution was measured. The cell density ({fraction (1/100)} dilution)reached an optical density of 0.215 at that time. A total of 175 gramsof dry dextrose was added and allowed to mix for 15 minutes, after which57.5 grams of acetophenone were added. Incubation was continued at 25°C., 300 rpm and 0.25 v/v/m aeration. Conversion was monitored by GC. Thereaction taking place is as follows:

[0184] D. Analysis:

[0185] Samples of broth were analyzed periodically by GC.

[0186] E. Product Recovers:

[0187] At the conclusion of the fermentation, the broth was pasteurizedfor 45 minutes at 65° C., cooled and stored in a refrigerator. The crudeproduct is recovered by extraction of the whole broth using an equalvolume of ethyl acetate. The spent broth is extracted a second time, andthe recovered ethyl acetate layers are combined and washed two timeswith saturated aqueous NaCl. The solvent is then evaporated to about 8%using a rotary evaporator with a water bath temperature of 40° C. and avacuum of 90-100 Torr. The crude extract is then fractionally distilledfor use for its organoleptic properties as exemplified, infra; or it isused for conversion to styralyl acetate with or without any furtherpurification. The resulting distilled product has a boiling point atatmospheric pressure of 204° C.; a melting point of 19.5-20.5° C.; aspecific gravity of 15° C. of 1.013; and a refractive index at 20° C. of1.5270.

[0188] NMR, GLC, chiral column GC and mass spectral analysis yield theinformation that the resulting product is a mixture of stereoisomershaving the structures:

[0189] having an optical rotation of −38.6°, [α=−38.6°] and anenantiomeric excess percent of about 87.5, [∈∈%=87.5±0.5].

EXAMPLE II Production of Styralyl Alcohol in Pilot Plant Fermenters

[0190] Reaction:

[0191] I. Inoculum Preparation

[0192] A. Medium:

[0193] The following medium is prepared by admixing the followingingredients: g/L Ingredients 5.0 AMBERFERM ® 4000 Peptone 5.0 TASTONE ®900 (yeast extract) 5.0 Malt extract 102 5.0 KH₂PO₄ (potassiumphosphate, monobasic) 0.5 MgSO₄.7H₂O (magnesium sulfate, heptahydrate) 1L Deionized water 1.0 Antifoam Hi MARS-010 ® FGK 50.0  CERELOSE ® 2001(added after sterilization) (dextrose)

[0194] B. Parameters: Temperature: 25° C.; Agitation: 100 rpm; andDuration: 24 hours.

[0195] A Fernbach flask containing 1 liter of inoculum medium wassterilized for 30 minutes at 121° C. The flask was inoculated with 1.8ml of a frozen culture of Kluyveromyces polysporus ATCC 22028; and 100ml of sterile 50% dextrose solution were added. The flask was incubatedin an incubator shaker (100 rpm) at 25° C. for 24 hours.

[0196] II. Seed Culture Preparation

[0197] A. Medium:

[0198] The following medium was prepared by admixing the followingingredients: g/L Ingredients 100.0 AMBERFERM ® 4000 Peptone 100.0TASTONE ® 900 (yeast extract) 100.0 Malt extract 102 100.0 KH₂PO₄(potassium phosphate, monobasic)  10.0 MgSO₄.7H₂O (magnesium sulfate,heptahydrate) 20.0 L Deionized water  10.0 Antifoam Hi MARS-010 ® FGK1,000.0   CERELOSE ® 2001 (added after sterilization)

[0199] B. Parameters: Broth Volume: 20 liters; Temperature: 25° C.;Agitation: 100 rpm; Aeration: 0.1 v/v/m (2 liters/minute); Duration: 24hours.

[0200] 20 Liters of inoculum medium were sterilized for 30 minutes in apilot plant 41 liter fermenter. After cooling, the fermenter wasinoculated with 1 liter of cells grown for a period of 24 hours and 2liters of sterile 50% dextrose were added. The fermenter was run at 25°C., 100 rpm and 0.1 v/v/m aeration for 24 hours. The pH is maintained inthe range of from about 5.8 up to about 6.

[0201] III. Production

[0202] A. Medium:

[0203] The following medium was prepared by admixing the followingingredients: g/L Ingredients 10.0  AMBERFERM ® 4000 Peptone 6.0TASTONE ® 900 (yeast extract) 7.5 Malt extract 102 5.0 KH₂PO₄ (potassiumphosphate, monobasic) 0.5 MgSO₄.7H₂O (magnesium sulfate, heptahydrate)1.0 L Deionized water 1.0 Antifoam Hi MARS-010 ® FGK 56.6  CERELOSE ®2001 (added after sterilization) 30.0  CERELOSE ® 2001 (added afteracetophenone) 38.0  Inoculum 1.5 NaOH (sodium hydroxide) 5.0Acetophenone 2.1 phosphoric acid 85%

[0204] B. Parameters: Inoculum: 20 liters of propagation culture; BrothVolume: 530 liters; Temperature: 25° C.; Aeration: 0.25 v/v/m;Agitation: 50 rpm; and Duration: 40 hours.

[0205] C. Process:

[0206] 530 Liters of production medium were sterilized in the pilotplant 300 gallon fermenter for 30 minutes at 121° C. After sterilizationand cooling, 60 liters of sterile 50% CERELOSE® dextrose and 20 litersof 24 hours-grown seed culture were added. The pH is maintained in therange of from about 5.5 up to about 6. After 15.5 hours of incubation,when the optical density is at 600 of a {fraction (1/100)} brothdilution was 0.171, a total of 15.9 kg dry dextrose powder was added.After 15 minutes of mixing, a total of 2.65 kg of acetophenone wasadded, and the agitation was increased to 100 rpm.

[0207] At the conclusion of the 24 hour incubation period, the broth iscooled for a period of 24 hours to room temperature and then pasteurizedfor 45 minutes at 65° C. The broth is then acidified to a pH in therange of 4-4.5 using 85% phosphoric acid, centrifuged and stored.

[0208] D. Product Recovery:

[0209] The broth is then extracted with ethyl acetate, and the ethylacetate extract is evaporated. The resulting oil is then fractionallydistilled at 204° C. and 1 atmosphere pressure to yield a product havinga specific gravity of 1.013 and a refractive index at 20° C. of 1.5270.

[0210] The resulting product is a mixture of stereoisomers having thestructures:

[0211] having an optical rotation of −38.6°, [α=−38.6°] and anenantiomeric excess percent of about 87.5, [∈∈%=87.5±0.5].

EXAMPLE III Production of Styralyl Acetate

[0212] Reaction:

[0213] Into a 2 liter reaction vessel equipped with stirrer,thermometer, reflux condenser and additional funnel are placed 118 gramsof the crude ethyl acetate extract of Example I, containing 85 grams ofstyralyl alcohol a mixture of stereoisomers having the structures:

[0214] 212.5 grams of acetic acid and 0.3 grams of citric acid wasrefluxed for 15 hours.

[0215] During the refluxing, 168 grams of acetic acid were recovered.

[0216] The refluxing conditions are 118-120° C. at 5 mm/Hg pressure.

[0217] The total of 130 grams crude is recovered and fractionallydistilled on a 2 inch×24 inch Goodloe column. Fractions 9-12 are bulkedyielding a mixture of stereoisomers having the structures:

[0218] having an optical rotation of −102.7°, [α=−102.7] and anenantiomeric excess percent of about 78.31, [∈∈%=78.31±0.5].

EXAMPLE IV Preparation of Styralyl Acetate

[0219] Reaction:

[0220] Into a 500 ml flask equipped with stirrer thermometer, refluxcondenser and addition funnel are placed 134 grams of the styralylalcohol mixture of stereoisomers prepared according to Example II; 0.5grams water and 3.25 grams NOVOZYM 435, an ester-forming enzyme which isCandida antarctica esterase expressed in Asperigilus orzae, atriacylglycerol hydrolase (E.C. No. 3.1.1.3) acting as an effectivecarboxylesterase (trademark of the Novo Nordisk A/S Organization of NovoAlle, 2880 Bagsvaerd, Denmark).

[0221] The resulting mixture with stirring is heated to 40° C. and overa period of 8 hours at a rate of 7.5 grams per hour, 60 grams of aceticacid is added to the reaction mass.

[0222] The reaction mass is continued to be stirred for a period of 48hours while maintaining the temperature at 40° C.

[0223] At the end of the 48 hour period, the resulting product isextracted with ethyl acetate and the ethyl acetate extract isconcentrated and then fractionally distilled yielding a product, asconfirmed by NMR, IR, mass spectral analysis and chiral GC analysis as amixture of stereoisomers having the structures:

[0224] having an optical rotation of +73.06°, [α=+73.06°] and anenantiomeric excess percent of about 81.1, [∈∈%=81.1±0.5].

[0225] The resulting product has a boiling point at atmospheric pressureof 213° C. a specific gravity at 15° C. of 1.024 and a refractive indexat 20° C. of 1.4948.

[0226] The conversion is 3.12%.

[0227] When the above example is carried out using in addition to theaforementioned reagents, 134 grams of n-hexane solvent, the conversionafter 48 hours increases to 9.1%.

EXAMPLE V Chypre Fragrance Formulations

[0228] The following Chypre formulations are prepared: Parts by WeightIngredients V(A) V(B) V(C) Musk ambrette 40 40 40 Musk ketone 60 60 60Coumarin 30 30 30 Oil of bergamot 150 150 150 Oil of lemon 100 100 100Methyl ionone 50 50 50 Hexyl cinnamic aldehyde 100 100 100Hydroxycitronellal 100 100 100 Oil of lavender 50 50 50 Texas cedarwoodoil 85 85 85 Virginia cedarwood oil 30 30 30 Oil of sandalwood(EastIndies) 40 40 40 Isoeugenol 20 20 20 Eugenol 10 10 10 Benzyl acetate 3030 30 beta-Phenyl ethyl alcohol 40 40 40 alpha-phenyl ethyl alcohol 3030 30 Oakmoss absolute 30 30 30 Vetiver oil of Venezuela 25 25 25 Themixture of stereoisomers defined according to 25 0 0 the structures:

prepared according to Example I, optical rotation −38.6°, [α = −38.6°]with an enantiomeric excess percent of about 87.5, [∈ ∈ % = 87.5 ± 0.5].The mixture of stereoisomers having the structures: 0 25 0

prepared according to Example III, having an optical rotation of−102.7°, [α = −102.7°] with an enantiomeric excess percent of about78.31, [∈ ∈ % = 78.31 ± 0.5]. The mixture of stereoisomers preparedaccording to 0 0 25 Example IV having the structures:

having an optical rotation of 73.06°, [α = +73.06°] with an enantiomericexcess percent of about 81.1, [∈ ∈ % = 81.1 ± 0.5].

[0229] The mixture of styralyl alcohol stereoisomers prepared accordingto Example I imparts so this Chypre formulation hyacinth, gardeniaundertones with strawberry topnotes. Accordingly, the aroma of theChypre formulation of Example V(A) is described as: “a Chypre aroma withhyacinth and gardenia undertones and strawberry topnotes.”

[0230] The mixture of styralyl acetate stereoisomers of Example IIIimparts to this Chypre formulation fresh strawberry, green, dried fruitundertones with green, strawberry topnotes. Accordingly, the aroma ofthe Chypre formulation of Example V(B) is described as: “a Chypre aromawith strawberry, green and dried fruit undertones and green, strawberrytopnoles.”

[0231] The mixture of styralyl acetate stereoisomers of Example IVimparts to the Chypre formulation of Example V(C) fruity, floral,mimosa, jasmine and gardenia undertones with fruity, strawberrytopnotes. Accordingly, the aroma of the Chypre formulation of ExampleV(C) is described as: “a Chypre aroma with fruity, floral, mimosa,jasmine and gardenia undertones and strawberry, fruity topnotes.”

EXAMPLE VI Preparation of a Cosmetic Powder Composition

[0232] Cosmetic powder compositions are prepared by mixing in a ballmill 100 grams of talcum powder with 0.25 grams of each of thesubstances set forth in Table II below. Each of the cosmetic powdercompositions has an excellent aroma as described in Table II below.TABLE II Substance Aroma Description The mixture of stereoisomers havingthe structures: A hyacinth, gardenia aroma with strawberry topnotes.

prepared according to Example I, having an optical rotation of −38.6°,[α = −38.6°] with an enantiomeric excess percent of about 87.5, [∈ ∈ % =87.5 ± 0.5]. The mixture of stereoisomers having the structures: Astrawberry, green, dried fruit aroma with green, strawberry topnotes.

prepared according to Example III, having an optical rotation of−102.7°, [α = −102.7°] with an enantiomeric excess percent of about78.31, [∈ ∈ % = 78.31 ± 0.5]. A mixture of stereoisomers having thestructures: A fruity, floral, jasmine, mimosa, gardenia aroma withfruity and strawberry topnotes.

having an optical rotation of +73.06°, [α = +73.06°] with anenantiomeric excess percent of about 81.1, [∈ ∈ % = 81.1 ± 0.5] preparedaccording to Example IV. The perfume composition of Example V(A). AChypre aroma with hyacinth and gardenia undertones and strawberrytopnotes. The perfume composition of Example V(B). A Chypre aroma withstrawberry, green and dried fruit undertones and green, strawberrytopnotes. The perfume composition of Example V(C). A Chypre aroma withfruity, floral, mimosa, jasmine and gardenia undertones and strawberry,fruity topnotes.

EXAMPLE VII Perfumed Liquid Detergent

[0233] Concentrated liquid detergents (lysine salt of n-dodecylbenzenesulfonic acid as more specifically described in U.S. Pat. No. 3,948,818issued on Apr. 6, 1976, incorporated by reference herein) with aromanuances as set forth in Table II of Example VI are prepared by adding0.10%, 0.15%, 0.20%, 0.25%, 0.30% and 0.35% of the substance set forthin Table II of Example VI. They are prepared by adding and homogeneouslymixing the appropriate quantity of substances set forth in Table II ofExample VI in the liquid detergent. The detergents all possess excellentaromas as set forth in Table II of Example VI, the intensity increasingwith greater concentrations of substance as set forth in Table II ofExample VI.

EXAMPLE VIII Preparation of a Colognes and Handkerchief Perfumes

[0234] Compositions as set forth in Table II of Example VI areincorporated into colognes at concentrations of 2.0%, 2.5%, 3.0%, 3.5%,4.0% and 5.0% in 80%, 85%, 90% and 95% aqueous food grade ethanolsolutions; and into handkerchief perfumes at concentrations of 15%, 20%,25% and 30% (in 80%, 85%, 90% and 95% aqueous food grade ethanolsolution). Distinctive and definite fragrances as set forth in Table IIof Example VI are imparted to the colognes and to the handkerchiefperfumes at all levels indicated.

EXAMPLE IX Preparation of a Soap Composition

[0235] 100 Grams of soap chips (per sample) (IVORY®, produced by theProcter & Gamble Company of Cincinnati, Ohio) are each mixed with 1 gramsamples of substances as set forth in Table II of Example VI untilhomogenous compositions are obtained. In each of the cases, thehomogeneous compositions are heated under 8 atmospheres pressure at 180°C. for a period of 3 hours and the resulting liquids are placed intosoap molds. The resulting soap cakes, on cooling, manifest aromas as setforth in Table II of Example VI.

EXAMPLE X Preparation of Solid Detergent Compositions

[0236] Detergents are prepared using the following ingredients accordingto Example I of Canadian Patent No. 1,007,948 (incorporated by referenceherein): Parts by Ingredients Weight NEODOL ® 45-11 (a C₁₄-C₁₅ alcoholethoxylated with 11 12 moles of ethylene oxide) Sodium carbonate 55Sodium citrate 20 Sodium sulfate. water brighteners q.s.

[0237] The detergent is a phosphate-free detergent. Samples of 100 gramseach of this detergent are admixed with 0.10, 0.15, 0.20 and 0.25 gramsof each of the substances as set forth in Table II of Example VI. Eachof the detergent samples has an excellent aroma as indicated in Table IIof Example VI.

EXAMPLE XI

[0238] Utilizing the procedure of Example I at column 15 of U.S. Pat.No. 3,632,396 (the disclosure of which is incorporated by referenceherein), a non-woven cloth substrate useful as drier-added fabricsoftening articles of manufacture are prepared wherein the substrate,substrate coating, outer coating and the perfuming material are asfollows:

[0239] (1) a water “dissolvable” paper (“Dissolve Paper”);

[0240] (2) ADOGEN 448 (melting point about 140° F.) as the substratecoating; and

[0241] (3) an outer coating having the following formulation (meltingpoint about 150° F.):

[0242] 57% C₂₀-C₂₂ HAPS;

[0243] 22% isopropyl alcohol;

[0244] 20% antistatic agent; and

[0245] 1% of one of the perfume materials as set forth in Table II ofExample VI.

[0246] Fabric softening compositions, prepared according to Example I atcolumn 15 of U.S. Pat. No. 3,632,396 having aroma characteristics as setforth in Table II of Example VI, consist of a substrate coating having aweight of about 3 grams per 100 square inches of substrate; a firstcoating on the substrate coating consisting of about 1.85 grams per 100square inches of substrate; and an outer coating coated on the firstcoating consisting of about 1.4 grams per 100 square inches ofsubstrate. One of the substances of Table II of Example VI is admixed ineach case with the outer coating mixture, thereby providing a totalaromatized outer coating weight ratio to substrate of about 0.5:1 byweight of the substrate. The aroma characteristics are imparted in apleasant manner to the headspace in a drier on operation thereof in eachcase, using the said drier-added fabric softener, non-woven fabrics, andthese aroma characteristics are described in Table II of Example VI.

EXAMPLE XII Hair Spray Formulations

[0247] The following hair spray formulation is prepared by firstdissolving PVP/VA E-735 copolymer manufactured by the GAF Corporation of140 West 51st Street, New York, N.Y. in 91.62 grams of 95% food gradeethanol. 8.0 Grams of the polymer is dissolved in the alcohol. Thefollowing ingredients are added to the PVP/VA alcoholic solution: WeightIngredients Percent Dioctyl sebacate 0.05 Benzyl alcohol 0.10 DowCorning 473 fluid prepared by the Dow Corning 0.10 Corporation TWEEN ®20 surfactant (prepared by ICI America 0.03 Corporation) One of theperfumery substances as set forth in Table II of 0.10 Example VI.

[0248] The perfuming substances as set forth in Table II of Example VIadd aroma characteristics as set forth in Table II of Example VI, whichare rather intense and aesthetically pleasing to the users of thesoft-feel, good-hold pump hair sprays.

EXAMPLE XIII Conditioning Shampoos

[0249] Monamid CMS (prepared by the Mona Industries Company) (3.0 weightpercent) is melted with 2.0 weight percent coconut fatty acid (preparedby the Procter & Gamble Company of Cincinnati, Ohio); 1.0 weight percentethylene glycol distearate (prepared by the Armak Corporation); andtriethanolamine (a product of the Union Carbide Corporation) (1.4 weightpercent). The resulting melt is admixed with Stepanol WAT produced bythe Stepan Chemical Company (35.0 weight percent). The resulting mixtureis heated to 60° C. and mixed until a clear solution is obtained (at 60°C.).

[0250] GAFQUAT® 755N polymer (manufactured by GAF Corporation of 140West 51st Street, New York, N.Y.) (5.0 weight percent) is admixed with0.1 weight percent sodium sulfite and 1.4 weight percent polyethyleneglycol 6000 distearate produced by the Armak Corporation.

[0251] The resulting material is then mixed and cooled to 45° C., and0.3 weight percent of perfuming substance as set forth in Table II ofExample VI is added to the mixture. The resulting mixture is cooled to40° C., and blending is carried out for an additional 1 hour in eachcase. At the end of this blending period, the resulting material has apleasant fragrance as indicated in Table II of Example VI.

EXAMPLE XIV

[0252] The following three tapioca pudding flavoring formulations areprepared: Parts by Weight XIV XIV XIV Ingredients (A) (B) (C)2-Methyl-2-pentenoic acid 5 5 5 2-Methyl-3-pentenoic acid 8 8 8 Ethylester of 2-methyl-3-pentenoic acid 4 4 4 n-Hexyl ester of2-methyl-4-pentenoic acid 6 6 6 The mixture of stereoisomers having thestructures: 2.5 0 0

prepared according to Example I, having an optical rotation −38.6°, [α =−38.6°] with an enantiomeric excess percent of about 87.5, [∈ ∈ % = 87.5± 0.5]. The mixture of stereoisomers having the structures: 0 2.5 0

prepared according to Example III, having an optical rotation of−102.7°, [α = −102.7°] with an enantiomeric excess percent of about78.31, [∈ ∈ % = 78.31 ± 0.5]. The mixture of stereoisomers preparedaccording to 0 0 2.5 Example IV having the structures:

having an optical rotation of +73.06°, [α = +73.06°] with anenantiomeric excess percent of about 81.1, [∈ ∈ % = 81.1 ± 0.5].

[0253] At the rate of 11 ppm, each of the above strawberry formulationsare added to separate portions of tapioca pudding formulations, and thetapioca pudding formulations are crafted into tapioca pudding portions.

[0254] The tapioca pudding portion of Example XIV(A) has an excellentstrawberry taste with fragrant strawberry jam aromas.

[0255] The tapioca pudding portion of Example XIV(B) has an excellentgreen, fresh strawberry taste with green, avocado and strawberry jamnuances.

[0256] The tapioca pudding portion of Example XIV(C) has an excellentstrawberry jam taste with apricot and apple flavor nuances.

What I is claimed is:
 1. A mixture of styralyl acetate optical isomershaving the structures:

having an optical rotation of +73.06°, [α=+73.06°] with an enantiomericexcess percent of about 81.1, [∈∈%=81.1±0.5].
 2. A process for preparinga mixture of styralyl alcohol optical isomers hating the structures:

having an optical rotation of −38.6°, [α=38.6°] with an enantiomericexcess percent of about 87.5, [∈∈%=87.5±0.5] comprising the steps ofcarrying out a fermentation of acetophenone in the presence of a cultureof Kluyveromyces polysporus ATCC 22028 according to the reaction:


3. A process for producing the stereoisomer mixture of claim 1comprising the step of admixing acetic acid and a mixture of styralylalcohol optical isomers having the structures:

having an optical rotation of −38.6°, [α=−38.6°] and an enantiomericexcess percent of about 87.5, [∈∈%=87.5±0.5], ill the presence of anester-forming enzyme according to the reaction:


4. The process of claim 3 wherein the ester-forming enzyme is Candidaantarctica esterase expressed in Asperigilus orzae, NOVOZYM® 435, atriacylglycerol hydrolase (E.C. No. 3.1.1.3) acting as an effectivecarboxylesterase.
 5. A mixture of styralyl alcohol optical isomershaving the structures:

having an optical rotation of −38.6°, [α=−38.6°] and an enantiomericexcess percent of about 87.5, [∈∈%=87.5±0.5].
 6. A mixture of styralylacetate optical isomers having the structures:

having an optical rotation of −102.7°, [α=−102.7°] and an enantiomericexcess percent of about 78.31, [∈∈%=78.31±0.5].
 7. A process forpreparing the stereoisomer composition of claim 6 comprising the step ofadmixing acetic acid with a mixture of styralyl alcohol optical isomershaving the structures:

having an optical rotation of −38.6°, [α=−38.6°] and an enantiomericexcess percent of about 87.5, [∈∈%=87.5±0.5] in the presence of a citricacid catalyst according to the reaction:


8. The process of claim 3 wherein the reaction is carried out in thepresence of an n-hexane solvent.
 9. The process of claim 3 wherein themixture of stereoisomers having the structures:

is extracted from the reaction mass with an ethyl acetate extractant.10. The process of claim 7 wherein the mixture of stereoisomers havingthe structures:

is extracted from the reaction mass with an ethyl acetate extractant.11. A process for augmenting or enhancing the aroma of a perfumecomposition, a cologne or a perfumed article comprising the step ofintimately admixing with a perfume base, a cologne base or a perfumedarticle base an aroma imparting, augmenting or enhancing quantity andconcentration of a composition of matter defined according to claim 1.12. A process for augmenting or enhancing the aroma of a perfumecomposition, a cologne or a perfumed article comprising the step ofintimately admixing with a perfume base, a cologne base or a perfumedarticle base an aroma imparting, augmenting or enhancing quantity andconcentration of a composition of matter defined according to claim 5.13. A process for augmenting or enhancing the aroma of a perfumecomposition, a cologne or a perfumed article comprising the step ofintimately admixing with a perfume base, a cologne base or a perfumedarticle base an aroma imparting, augmenting or enhancing quantity andconcentration of a composition of matter defined according to claim 6.14. A perfume composition comprising a perfume base and intimatelyadmixed therewith an aroma augmenting, enhancing or imparting quantityand concentration of the composition of matter of claim
 1. 15. A perfumecomposition comprising a perfume base and intimately admixed therewithan aroma augmenting, enhancing or imparting quantity and concentrationof the composition of matter of claim
 5. 16. A perfume compositioncomprising a perfume base and intimately admixed therewith an aromaaugmenting, enhancing or imparting quantity and concentration of thecomposition of matter of claim
 6. 17. A perfumed article comprising aperfumed article base and intimately admixed therewith an aromaaugmenting, enhancing or imparting quantity and concentration of thecomposition of matter defined according to claim
 1. 18. A perfumedarticle comprising a perfumed article base and intimately admixedtherewith an aroma augmenting, enhancing or imparting quantity andconcentration of the composition of matter defined according to claim 5.19. A perfumed article comprising a perfumed article base and intimatelyadmixed therewith an aroma augmenting, enhancing or imparting quantityand concentration of the composition of matter defined according toclaim
 6. 20. A cologne consisting of water, ethanol and an aromaimparting quantity of the composition defined according to claim
 1. 21.A cologne consisting of water, ethanol and an aroma imparting quantityof the composition defined according to claim
 5. 22. A cologneconsisting of water, ethanol and an aroma imparting quantity of thecomposition defined according to claim
 6. 23. A process for augmenting,enhancing or imparting a flavor in or to a foodstuff, chewing gum orbeverage comprising the step of intimately admixing with a foodstuffbase, a beverage base or a chewing gum base an aroma and tasteaugmenting, imparting or enhancing quantity and concentration of thecomposition of matter of claim
 1. 24. A process for augmenting,enhancing or imparting a flavor in or to a foodstuff, chewing gum orbeverage comprising the step of intimately admixing with a foodstuffbase, a beverage base or a chewing gum base an aroma and tasteaugmenting, imparting or enhancing quantity and concentration of thecomposition of matter of claim
 5. 25. A process for augmenting,enhancing or imparting a flavor in or to a foodstuff, chewing gum orbeverage comprising the step of intimately admixing with a foodstuffbase, a beverage base or a chewing gum base an aroma and tasteaugmenting, imparting or enhancing quantity and concentration of thecomposition of matter of claim
 6. 26. The process of claim 23 whereinthe foodstuff composition is a strawberry-flavored foodstuff.
 27. Theprocess of claim 24 wherein the foodstuff composition is astrawberry-flavored foodstuff.
 28. The process of claim 25 wherein thefoodstuff composition is a strawberry-flavored foodstuff.